ABSTRACT In conventional T cells, signaling pathways that control cellular metabolism have a crucial role in dictating the outcome of T cell activation and their effector function. Resting CD4 and CD8 T cells use predominantly oxidative metabolism but stimulation leads them to sharply increase glucose metabolism and adopt aerobic glycolysis as a primary metabolic program. However, little is known about metabolic regulation and its role for cellular functions of Natural Killer T (NKT) cells although recent studies indicate an important role of metabolism in NKT cell differentiation. NKT cells are a heterogeneous population that shows a high degree of phenotypic and functional specialization. When activated, unlike conventional T cells, NKT cells exhibit a fast and robust effector function such as cytokine release or cytotoxicity. NKT cells can exert either an inflammatory or a regulatory function depending on the tissue type such as liver or adipose tissue, respectively, that are a site of metainflammation. Thus, the metabolic regulation in NKT cells likely plays an important role in immune diseases. To understand how NKT cells regulate their metabolism to mediate an appropriate immune response under a different environment, we measured parameters that associate with the metabolic capacity and compared them with that of CD4 T cells. Our study revealed that NKT cells are very different from CD4 T cells in many ways. NKT cells rely more on oxidative phosphorylation for their survival and repurposed Glc carbon is used for optimal cytokine expression. In addition, NKT cells rely on glutamine (Gln) metabolism for proliferation but not to express IFN-?. Gln starved NKT cells seem to be inefficient to switch to glucose (Glc) metabolism, which is similar to Gln-addicted cancer cells. Metabolomic data revealed that NKT cells have elevated glutamine metabolites prior to stimulation further supporting an essential role of Gln metabolism. mTORC and AMPK is known to increase and decrease Gln metabolism, respectively. In line with this, NKT cells are sensitive to mTORC inhibition but AMPK deficiency results in hyperproliferation and increased cytokine expression. Based on our data, we hypothesize that the proper regulation of Gln metabolism is critical for NKT cells? survival and function, which is controlled by the balance between mTORC and AMPK activity. To test the hypothesis, we propose two specific aims. Aim 1 will investigate the mechanisms by which Gln controls NKT cell proliferation and function, and Aim 2 will focus on the mTORC-AMPK axis to discern the regulation of Gln metabolism in NKT cells. Metabolic status and regulation in NKT cells is virtually unexplored and, therefore, investigating the regulation of the metabolic networks in NKT cells is highly innovative. Undoubtedly, the proposed studies are significant given the fact that these studies will address a poorly understood area, and the results will establish specific and selective metabolic demands of NKT cells.